Biodegradable cationic polymers

ABSTRACT

Polymers comprising a polyethylenimine, a biodegradable group, and a relatively hydrophobic group are useful for the delivery of bioactive agents to cells.

This application is a continuation of U.S. application Ser. No.11/216,986, filed Aug. 31, 2005, now U.S. Pat. No. 7,358,223, whichclaims priority to U.S. Provisional Application No. 60/615,764, filedOct. 4, 2004 and U.S. Provisional Application No. 60/698,357, filed Jul.11, 2005, all of which are hereby incorporated by reference in theirentireties.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledNDTCO-037C1.TXT, created Jan. 14, 2008, which is 1 Kb in size. Theinformation in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to compositions and methods for deliveringbioactive agents to cells. In preferred embodiments, this inventionrelates to cationic lipopolymers comprising a polyethylenimine (PEI), abiodegradable group, and a relatively hydrophobic group, and to methodsof using such lipopolymers to deliver bioactive agents such as DNA, RNA,oligonucleotides, proteins, peptides, and drugs.

2. Description of the Related Art

A number of techniques are available for delivering bioactive agents tocells, including the use of viral transfection systems and non-viraltransfection systems. Viral systems typically have higher transfectionefficiency than non-viral systems, but there have been questionsregarding the safety of viral systems. See Verma I. M and Somia N.,Nature 389 (1997), pp. 239-242; Marhsall E. Science 286 (2000), pp.2244-2245. In addition, viral vector preparation tends to be acomplicated and expensive process. Although non-viral transfectionsystems generally are less efficient than viral systems, they havereceived significant attention because they are generally believed to besafer and easier to prepare than viral systems.

A number of non-viral transfection systems involve the use of cationicpolymers that are complexed to bioactive agents. Examples of cationicpolymer that have been used as gene carriers include poly(L-lysine)(PLL), polyethyleneimine (PEI), chitosan, PAMAM dendrimers, andpoly(2-dimethylamino)ethyl methacrylate (pDMAEMA). Unfortunately,transfection efficiency is typically poor with PLL, and high molecularweight PLL has shown significant toxicity to cells. In some cases PEIprovides efficient gene transfer without the need for endosomolytic ortargeting agents. See Boussif O., Lezoualc'h F., Zanta M. A., Mergny M.D., Scherman D., Demeneix B., Behr J. P., Proc Natl Acad Sci USA. Aug.1, 1995, 92(16) 7297-301. A range of polyamidoamine dendrimers have beenstudied as gene-delivery systems. See Eichman J. D., Bielinska A. U.,Kukowska-Latallo J. F., Baker J. R. Jr., Pharm. Sci. Technol. Today 2000July; 3(7):232-245. Unfortunately, both PEI and dendrimers have beenreported to be toxic to cells, thus limiting the potential for using PEIas a gene delivery tool in applications to human patients. In addition,the cost of polyamidoamine dendrimers having commercially practical genetransfection efficiencies is relatively high.

Gene carriers made with degradable cationic polymers have been reportedto transfer genes into mammalian cells with decreased cytotoxicity. SeeLim Y. B., Kim S. M., Lee Y., Lee W. K., Yang T. G., Lee M. J., Suh H.,Park J. S., J. Am. Chem. Soc., 123 (10), 2460-2461, 2001. Unfortunately,these degradable systems also exhibited lower gene transfer efficiencycompared to non-degradable polymers. To improve the transfectionefficiency of low molecular weight PEI, Gosselin et al. reported thathigher molecular weight PEI could be obtained by usingdisulfide-containing linkers. See Gosselin, Micheal A., Guo, Menjin, andLee, Robert J. Bioconjugate Chem. 2001. 12:232-245. PEI polymers madeusing dithiobis(succinimidylpropionate) (DSP) anddimethyl-3,3′-dithiobispropionimidate-2HCl (DTBP) showed comparable genetransfection efficiency and lower cytotoxicity. However, thedisulfide-containing linkers are expensive, which makes large-scalepreparation of this system difficult and undesirable. The polymers withdisulfide-containing linkers are only degraded under reducingconditions, which limits polymer applications in other conditions.

Lynn, et al. have described a method of synthesizing biodegradablecationic polymers using diacrylates as linker molecules between cationiccompounds. See Lynn, David A.; Anderson, Daniel G.; Putnam, David; andLanger, Robert. J. Am. Chem. Soc. 2001, 123, 8155-8156. However, theresulting polymers do not complex well with many bioactive agents.Synthesis of these polymers requires days to complete and the amount ofeffective product, which can be used in gene delivery, is low. More thanone hundred cationic polymers were produced according to the methods ofLynn et al., but only two of these polymers showed effective genetransfection efficiency. These factors make the preparation of highmolecular weight polymers by this method difficult to achieve.

Thus, there remains a need for cationic polymers that may be used tosafely and efficiently facilitate the delivery of bioactive agents tocells.

SUMMARY OF THE INVENTION

An embodiment provides a polymer comprising a recurring unit selectedfrom the group consisting of formula (Ia) and formula (Ib):

wherein: PEI is a polyethyleneimine recurring unit; R is selected fromthe group consisting of electron pair, hydrogen, C₂-C₁₀ alkyl, C₂-C₁₀heteroalkyl, C₅-C₃₀ aryl, and C₂-C₃₀ heteroaryl; L is selected from thegroup consisting of C₂-C₅₀ alkyl, C₂-C₅₀ heteroalkyl, C₂-C₅₀ alkenyl,C₂-C₅₀ heteroalkenyl, C₅-C₅₀ aryl; C₂-C₅₀ heteroaryl; C₂-C₅₀ alkynyl,C₂-C₅₀ heteroalkynyl, C₅-C₅₀ aryl; C₂-C₅₀ heteroaryl; C₂-C₅₀carboxyalkenyl, and C₂-C₅₀ carboxyheteroalkenyl; W is a cationic moietycomprising from about 2 to about 50 carbon atoms; and m is an integer inthe range of about 1 to about 30.

A recurring unit selected from the group consisting of formula (Ia) andformula (Ib) may be referred to herein as a recurring unit of theformula (I). Thus, for example, a polymer comprising a recurring unitselected from the group consisting of formula (Ia) and formula (Ib) maybe referred to herein as a polymer comprising a recurring unit of theformula (I) or as a polymer of the formula (I). A polymer comprising arecurring unit of the formula (I) may be referred to herein as acationic lipopolymer.

In preferred embodiments, the PEI in formulae (Ia) and (Ib) isrepresented by a recurring unit of formula (II)

wherein x is an integer in the range of about 1 to about 100 and y is aninteger in the range of about 1 to about 100. Those skilled in the artwill appreciate that the nitrogen atom in the recurring unit of formula(I) may bear a cationic charge and thus may form an ionic bond withvarious negatively charged species, e.g., with an anion such aschloride, bromide, iodide, sulfate, etc. Multiple polymers of theformula (I) may be prepared and stored until use, and thus anotherembodiment provides a polymer library comprising a plurality ofpolymers, each of the polymers individually comprising a recurring unitof the formula (I), wherein at least one parameter selected from thegroup consisting of R, L, PEI, W and m is different for at least two ofthe polymers.

In another embodiment, the polymer comprising a recurring unit of theformula (I) further comprises a biomolecule that is complexed to thepolymer. The biomolecule may bear one or more an anionic groups and mayform an ionic bond with the recurring unit of formula (I). Examples ofbiomolecules bearing one or more anionic groups include nucleic acids(e.g., DNA, single strand RNA, double strand RNA, ribozyme, DNA-RNAhybridizer, siRNA, antisense DNA and antisense oligo), proteins,peptides, lipids, and carbohydrates.

In another embodiment, the polymer comprising a recurring unit of theformula (I) further comprises a biomolecule, a delivery enhancing agentcapable of entering a eukaryotic cell, and/or a diagnostic imagingcomposition that is complexed to the polymer. The delivery enhancingagent may facilitate one or more functions in the eukaryotic cell, e.g.,receptor recognition, internalization, escape of the biomolecule fromcell endosome, nucleus localization, biomolecule release, and systemstabilization.

The polymer comprising a recurring unit of formula (I) which furthercomprises a biomolecule, and which may further comprise a deliveryenhancing agent capable of entering a eukaryotic cell and/or adiagnostic imaging composition that is complexed to the polymer, isuseful for transfecting eukaryotic cells. Thus, another embodimentprovides a method of transfecting a eukaryotic cell, comprisingcontacting the cell with such a polymer (comprising a recurring unit offormula (I) and a biomolecule, optionally further comprising a deliveryenhancing agent and/or a diagnostic imaging composition) to therebydeliver the biomolecule to the cell. The method may involve treating amammal, comprising identifying a mammal in need of gene therapy andadministering such a polymer to the mammal. In a preferred embodiment,the biomolecule is siRNA, wherein the siRNA is effective to lowerexpression of a gene of interest.

Another embodiment provides a medical diagnostic system comprising aligand that recognizes a specific receptor of a eukaryotic cell and apolymer, where the polymer comprises a recurring unit of formula (I) anda biomolecule, and may further comprise a delivery enhancing agentcapable of entering a eukaryotic cell and/or a diagnostic imagingcomposition that is complexed to the polymer.

Another embodiment provides a pharmaceutical composition comprising: asensitizer agent and a polymer, where the polymer comprises a recurringunit of formula (I) and a biomolecule, and may further comprise adelivery enhancing agent capable of entering a eukaryotic cell and/or adiagnostic imaging composition that is complexed to the polymer. Thesensitizer agent may be a compound that undergoes a change in propertieson exposure to light or other stimuli, thereby facilitating delivery ofthe biomolecule (e.g., by increasing the degradation rate of thepolymer). The sensitizer agent may itself be a biomolecule thatundergoes a change in activity upon stimulus.

These and other embodiments are described in greater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates typical GFP signals in 293 cells after transfectionby lipopolymers (5A, 5B and 5C) and lipofectamine 2000.

FIG. 2 illustrates typical GFP signals in 208 F cells after transfectionby lipopolymers (5A, 5B and 5C) and lipofectamine 2000.

FIG. 3 shows plots of the relative fluorescent level of 208 F and 293cells after transfection by lipopolymers (5A, 5B and 5C) andlipofectamine 2000 (“lipo”).

FIG. 4 illustrates typical GFP signals in HT-GFP cells after siRNAdelivery by lipopolymers (5A, 5B and 5C) and lipofectamine 2000.

FIG. 5 illustrates typical GFP signal in HeLa-GFP cells after siRNAdelivery by lipopolymers (5A, 5B and 5C) and lipofectamine 2000.

FIG. 6 shows plots of the relative fluorescent level of HT-GFP and HeLaGFP cells siRNA after delivery by lipopolymers (5A, 5B and 5C) andlipofectamine 2000 (“lipo”).

FIG. 7 shows a plot of the cell survival fraction of lipopolymers (5A,5B and 5C) and lipofectamine 2000 (“lipo”) after siRNA delivery.

FIG. 8 shows a plot of the relative fluorescent level of HT-GFP geneafter different SiRNA delivered by the cationic lipopolymers (5A, 5B and5C).

FIG. 9 shows a plot of the relative fluorescent unit of antisenseoligo/lipopolymers complexes compared with free oligo solution.

FIG. 10 illustrates gel electrophoresis results for binding of polymer 6to DNA.

FIG. 11 illustrates typical GFP signals of 293 cells after transfectionby 7A and lipofectamine 2000.

FIG. 12 shows a plot of Luciferase activity in luc 705 cell afterantisense oligo delivery by polymer 7A and by lipofectamine 2000.

FIG. 13 shows a plot of Luciferase activity in CHO-AA8 luc after polymer7A and lipofectamine 2000 mediated SiRNA delivery.

FIG. 14 shows a plot illustrating cell survival fraction aftertransfection using 7A and lipofectamine.

FIG. 15 shows a plot of GFP transfection efficiency of 7A afterincubation in opti MEM for various periods of time.

DETAILED DESCRIPTION OF THE EMBODIMENTS

An embodiment provides cationic lipopolymers comprising apolyethylenimine, a biodegradable group, and a relatively hydrophobic“lipo” group. Preferred cationic lipopolymers comprise a recurring unitselected from the group consisting of formula (Ia) and formula (Ib):

It will be understood that the “formula (I)” as used herein refers toboth formula (Ia) and formula (Ib). In formula (I), PEI ispolyethyleneimine (e.g., a polymer comprising recurring unitsrepresented by the formula (II) above), the ester linkages arebiodegradable groups, L represents a relatively hydrophobic “lipo”group, and m is in the range of about 1 to about 30. For example, incertain embodiments L is selected from the group consisting of C₂-C₅₀alkyl, C₂-C₅₀ heteroalkyl, C₂-C₅₀ alkenyl, C₂-C₅₀ heteroalkenyl, C₅-C₅₀aryl; C₂-C₅₀ heteroaryl; C₂-C₅₀ alkynyl, C₂-C₅₀ heteroalkynyl, C₅-C₅₀aryl; C₂-C₅₀ heteroaryl; C₂-C₅₀ carboxyalkenyl, and C₂-C₅₀carboxyheteroalkenyl. In preferred embodiments, L is selected from thegroup consisting of C₁₂ to C₁₈ fatty acid, cholesterol, and derivativesthereof. The R in formula (I) may represent an electron pair or ahydrogen atom. Those skilled in the art understand that when Rrepresents an electron pair, the recurring unit of formula (I) iscationic at low pH. The R in formula (I) may also represent a relativelyhydrophobic lipo group such as C₂-C₁₀ alkyl, C₂-C₁₀ heteroalkyl, C₅-C₃₀aryl, or C₂-C₃₀ heteroaryl, in which case it will be understood that thenitrogen atom bears a cationic charge, generally over a wide pH range.

Cationic lipopolymers comprising a recurring unit of formula (I) may beprepared by reacting a diacrylate monomer of the formula (III) with apolyethyleneimine (PEI) as shown in Scheme A below:

In formula (III), R and L have the same meanings as described above forcationic lipopolymers comprising a recurring unit of formula (I). SchemeA illustrates the preparation of a polymer comprising a recurring unitof the formula (Ia). It will be understood that a polymer comprising arecurring unit of the formula (Ib) may be prepared in a similar mannerfrom the appropriate W group-containing diacrylate monomer. The PEIpreferably contains recurring units of the formula (II) in which x is aninteger in the range of about 1 to about 100 and y is an integer in therange of about 1 to about 100.

The reaction illustrated in Scheme A may be carried out by intermixingthe PEI and the diacrylate (III) in a mutual solvent such as ethanolwith stirring, preferably at room temperature for several hours, thenevaporating the solvent to recover the resulting polymer. This inventionis not bound by theory, but it is believed that the reaction between thePEI and diacrylate (III) involves a Michael reaction between one or moreamines of the PEI with double bond(s) of the diacrylate. See J. March,Advanced Organic Chemistry 3^(rd) Ed., pp. 711-712 (1985). Thediacrylate shown in Scheme A may be prepared in the manner illustratedin the Examples described below.

A wide variety of polymers comprising a recurring unit of the formula(I) may be made in accordance with Scheme A by varying the molecularweight and structure of the PEI, the size and type of the R, W and Lgroups on the diacrylate (III), and the ratio of diacrylate (III) toPEI. Mixtures of different diacrylates and/or mixtures different PEI'smay be used. The PEI may be multifunctional, and thus may be capable ofreacting with two or more diacrylates. Crosslinking agents may be usedto produce a crosslinked cationic lipopolymer and/or the relativeproportions of multifunctional PEI and diacrylate (II) may be adjustedto produce a crosslinked cationic lipopolymer. The molecular weight ofthe PEI is preferably in the range of about 600 to about 25,000 daltons.The molar ratio of PEI to diacrylate is preferably in the range of about1:2 to about 1:20. The weight average molecular weight of the cationiclipopolymer may be in the range of about 500 Daltons to about 1,000,000Daltons preferably in the range of about 2,000 Daltons to about 200,000Daltons. Molecular weights may be determined by size exclusionchromatography using PEG standards or by agarose gel electrophoresis. Inan embodiment, a polymer library is provided by preparing a plurality ofcationic lipopolymers in which R, L, W, PEI, and/or m are different forat least two of the polymers.

The cationic lipopolymer is preferably degradable, more preferablybiodegradable, e.g., degradable by a mechanism selected from the groupconsisting of hydrolysis, enzyme cleavage, reduction, photo-cleavage,and sonication. This invention is not limited by theory, but it isbelieved that degradation of the cationic lipopolymer of formula (I)within the cell proceeds by enzymatic cleavage and/or hydrolysis of theester linkages.

The cationic lipopolymers may form complexes with biomolecules and thusare useful as carriers for the delivery of biomolecules to cells.Examples of biomolecules that form complexes with cationic lipopolymersof the formula (I) include nucleic acids, proteins, peptides, lipids,and carbohydrates. Examples of nucleic acids include DNA, single strandRNA, double strand RNA, ribozyme, DNA-RNA hybridizer, and antisense DNA,e.g., antisense oligo. A preferred nucleic acid is siRNA. Cationiclipopolymers that comprise a biomolecule that is complexed to thepolymer may be formed by intermixing the cationic lipopolymers andbiomolecules in a mutual solvent, more preferably by the methodsdescribed in the examples below.

Cationic lipopolymers that comprise a biomolecule that is complexed tothe polymer may further comprise a delivery enhancing agent capable ofentering a eukaryotic cell. The delivery enhancing agent may bedissolved or mixed with the complex, or may be coupled (e.g., covalentlybonded or complexed) to the cationic lipopolymer. Delivery enhancers aresubstances that facilitate transport of a biomolecule into a cell,typically by enhancing transport of a biomolecule/carrier complex acrossa membrane, reducing degradation during transport, and/or facilitatingrelease of the biomolecule from the carrier. Transport of a biomolecule,such as a gene, into a cell preferably involves releasing thebiomolecule from the carrier after the biomolecule/carrier complex hascrossed the cell membrane, endosome membrane, and nuclear membrane. Forexample, in the case of a nucleic acid, the nucleic acid/carrier complexfirst passes through the cell membrane. When this is accomplished byendocytosis, the nucleic acid/carrier complex is then internalized. Thecarrier along with the nucleic acid-cargo is enveloped by the cellmembrane by the formation of a pocket and the pocket is subsequentlypinched off. The result is a cell endosome, which is a largemembrane-bound structure enclosing the nucleic acid cargo and thecarrier. The nucleic acid-carrier complex then escapes through theendosome membrane into the cytoplasm, avoiding enzyme degradation in thecytoplasm, and crosses the nuclear membrane. Once in the nucleus, thenucleic acid cargo separates from the carrier.

In general, delivery enhancers fall into two categories: viral carriersystems and non-viral carrier systems. Because human viruses haveevolved ways to overcome the barriers to transport into the nucleusdiscussed above, viruses or viral components are useful for transportingnucleic acids into cells. One example of a viral component useful as adelivery enhancer is the hemagglutinin peptide (HA-peptide). This viralpeptide facilitates transfer of biomolecules into cells by endosomedisruption. At the acidic pH of the endosome, this protein causesrelease of the biomolecule and carrier into the cytosol. Other examplesof viral components useful as delivery enhancers are known to thoseskilled in the art.

Non-viral delivery enhancers are typically either polymer-based orlipid-based. They are generally polycations which act to balance thenegative charge of the nucleic acid. Polycationic polymers have shownsignificant promise as non-viral gene delivery enhancers due in part totheir ability to condense DNA plasmids of unlimited size and to safetyconcerns with viral vectors. Examples include peptides with regions richin basic amino acids such as oligo-lysine, oligo-arginine or acombination thereof and PEI. These polycationic polymers are believed tofacilitate transport by condensation of DNA. Branched chain versions ofpolycations such as PEI and starburst dendrimers can mediate both DNAcondensation and endosome release. See Boussif, et al. (1995) Proc.Natl. Acad. Sci. USA vol. 92: 7297-7301. PEI can be prepared as a highlybranched polymer with terminal amines that are ionizable at pH 6.9 andinternal amines that are ionizable at pH 3.9. Because of thisorganization, PEI can generate a change in vesicle pH that leads tovesicle swelling and, eventually, release from endosome entrapment.

Another way of enhancing delivery is for the cationic lipopolymer tocomprise a ligand that is recognized by a receptor on the cell that hasbeen targeted for biomolecule cargo delivery. Biomolecule delivery intothe cell may then be initiated by receptor recognition. In this context,the term “ligand” refers to a biomolecule which can bind to a specificreceptor protein located on the surface of the target cell or in itsnucleus or cytosol. In an embodiment, the ligand may be an antibody,hormone, pheromone, or neurotransmitter, or any biomolecule capable ofacting like a ligand, which binds to the receptor. An antibody refers toany protein produced by a B lymphocyte in response to an antigen. Whenthe ligand binds to a particular cell receptor, endocytosis isstimulated. Examples of ligands which have been used with various celltypes to enhance biomolecule transport are galactose, transferrin, theglycoprotein asialoorosomucoid, adenovirus fiber, malariacircumsporozite protein, epidermal growth factor, human papilloma viruscapsid, fibroblast growth factor and folic acid. In the case of thefolate receptor, the bound ligand is internalized through a processtermed potocytosis, where the receptor binds the ligand, the surroundingmembrane closes off from the cell surface, and the internalized materialthen passes through the vesicular membrane into the cytoplasm. SeeGottschalk, et al. (1994) Gene Ther 1:185-191.

Various delivery enhancing agents are believed to function by endosomedisruption. For example, in addition to the HA-protein described above,defective-virus particles have also been used as endosomolytic agents.See Cotten, et al. (July 1992) Proc. Natl. Acad. Sci. USA vol. 89: pages6094-6098. Non-viral agents are typically either amphiphillic orlipid-based.

The release of biomolecules such as DNA into the cytoplasm of the cellmay be enhanced by agents that mediate endosome disruption, decreasedegradation, or bypass this process all together. Chloroquine, whichraises the endosomal pH, has been used to decrease the degradation ofendocytosed material by inhibiting lysosomal hydrolytic enzymes. SeeWagner, et al. (1990) Proc Natl Acad Sci USA vol. 87: 3410-3414.Branched chain polycations such as PEI and starburst dendrimers alsopromote endosome release as discussed above.

Endosomal degradation may be bypassed by incorporating subunits oftoxins such as Diptheria toxin and Pseudomonas exotoxin as components ofchimeric proteins that may be incorporated into the cationiclipopolymer/biomolecule complex. See Uherek, et al.(1998) J. Biol. Chem.vol. 273: 8835-8841. These components promote shuttling of the nucleicacid through the endosomal membrane and back through the endoplasmicreticulum.

Once in the cytoplasm, transport of the biomolecule cargo to the nucleusmay be enhanced by inclusion of a nuclear localization signal on thenucleic acid-carrier. For example, a specific amino acid sequence thatfunctions as a nuclear-localization signal (NLS) may be used. It isbelieved that the NLS on a biomolecule/carrier complex interacts with aspecific nuclear transport receptor protein located in the cytosol. Oncethe biomolecule/carrier complex is assembled, the receptor protein inthe complex is thought to make multiple contacts with nucleoporins,thereby transporting the complex through a nuclear pore. After thebiomolecule/carrier complex reaches its destination, it dissociates,freeing the cargo and other components. The sequencePro-Lys-Lys-Lys-Arg-Lys-Val (SEQ ID NO.: 1) from the SV40 largeT-antigen may be used for transport into nuclei. It is believed thatthis short sequence from SV40 large T-antigen may provide a signal thatcauses the transport of associated macromolecules into the nucleus.

The cationic lipopolymer may further comprise a diagnostic imagingcompound such as a fluorescent, radioactive, or radio-opaque dye that iscomplexed to the polymer. The complex may be formed by intermixing thecationic lipopolymer and the diagnostic imaging compound in a mutualsolvent. After administration to a mammal, the polymer (complexed withthe diagnostic imaging compound) may be tracked using well knowntechniques such as PET, MRI, CT, SPECT, etc. (see Molecular Imaging ofGene Expression and Protein Function In Vivo With PET and SPECT, VijaySharma, PhD, Gary D. Luker, MD, and David Piwnica-Worms, MD, Ph.D.,JOURNAL OF MAGNETIC RESONANCE IMAGING 16:336-351 (2002)).

Another embodiment provides a pharmaceutical composition comprising: asensitizer agent and a polymer, where the polymer comprises a recurringunit of formula (I) and a biomolecule, and may further comprise adelivery enhancing agent capable of entering a eukaryotic cell and/or adiagnostic imaging composition that is complexed to the polymer. Thesensitizer agent may be a compound that undergoes a change in propertieson exposure to light or other stimuli, thereby facilitating delivery ofthe biomolecule (e.g., by increasing the degradation rate of thepolymer). The sensitizer agent may itself be a biomolecule thatundergoes a change in activity upon stimulus. The sensitizer agent maybe a light activated drug. Suitable light activated drugs include, butare not limited to, fluorescein, merocyanin, xanthene and itsderivatives and the photoreactive pyrrole-derived macrocycles and theirderivatives. Suitable photoreactive pyrrole-derived macrocycles include,but are not limited to, naturally occurring or synthetic porphyrins,naturally occurring or synthetic chlorins, naturally occurring orsynthetic bacteriochlorins, synthetic isobateriochlorins,phthalocyanines, naphtalocyanines, and expanded pyrrole-basedmacrocyclic systems such as porphycenes, sapphyrins, and texaphyrins.

EXAMPLE 1

Oxalyl chloride (13.5 mL, 152 mmol) was added to a solution of oleicacid 1 (10.7 g, 38 mmol) in dichloromethane (DCM, 200 mL) andN,N-dimethylformamide (DMF, three drops) at 0° C. The reaction mixturewas stirred for about 1 hour and then allowed to warm to roomtemperature. After 1 hour, the solution was diluted with toluene anddistilled. The residue was dissolved in dichloromethane (200 mL) andcooled to 0° C. Diethanolamine (10.9 mL, 114 mmol),4-(dimethylaminopyridine (490 mg, 4 mmol), and triethylamine (21 mL, 152mmol) were added to the solution. The solution was stirred at 0° C. for30 minutes and then allowed to proceed at room temperature overnight.The reaction mixture was diluted with dichloromethane and washed with 1N HCl and aqueous NaHCO₃. The organic phase was dried (Na₂SO₄) andconcentrated under reduced pressure. The crude residue was then purifiedon a silica gel column (10:1 ethyl acetate: methanol), yielding 13.5 g(99.9%) of compound 2 as a colorless oil.

EXAMPLE 2

Triethylamine (8.1 g, 80 mmol), DMAP (0.5 g, 4 mmol) and 2 (7.1 g, 20mmol) was dissolved in 200 ml of dichloromethane at room temperature.The system was flushed with argon and the solution was cooled in an icebath. Acryolyl chloride (5.4 g, 60 mmol) in 25 ml of dichloromethane wasadded dropwise. After the addition the reaction was allowed to warm toroom temperature and stir overnight. The reaction mixture was dilutedwith dichloromethane and washed with water and aqueous NaHCO₃. Theorganic phase was dried (Na₂SO₄) and concentrated under reducedpressure. The crude residue was then purified on a silica gel column(1:3 ethyl acetate:hexane), yielding 7.5 g (81%) of compound 3 as acolorless oil.

EXAMPLE 3

The synthesis of a cationic lipopolymer was carried out in accordancewith Scheme A by reacting polyethylenimine having a molecular weight of600 (PEI-600) with compound 3 as follows: About 0.6 g of PEI-600(Aldrich) was weighed and placed in a small vial, and 10 ml of ethanolwas added. After the PEI-600 completely dissolved, 3.7 g (8.0 mmol) of 3in 10 ml of ethanol was added quickly into the PEI solution whilestirring. The reaction mixture was stirred for 2 hours at roomtemperature. After removing the organic solvent under reduced pressure,a transparent, viscous liquid was obtained. ¹H-NMR spectrum indicatedthat the acrylic carbon-carbon double bond disappeared completely. Themolecular weight of the obtained polymer was estimated by agarose gelelectrophoresis. This is a general procedure that serves as a model forother synthetic procedures involving similar compounds, and may be usedto synthesize a series of degradable cationic lipopolymers.

EXAMPLE 4

A cationic lipopolymer was prepared as described in Example 3 exceptthat, after stirring the reaction mixture for 2 hours at roomtemperature (25° C.), the reaction mixture is neutralized by adding 2.5ml of 4M HCl in ether. The white precipitate 5C is filtered, washed withethanol, and dried at room temperature under reduced pressure. Theobtained polymer 5C was characterized with NMR spectrometer and agarosegel electrophoresis. Polymers 5A and 5B were prepared in a similarmanner: 5A: PEI₁₈₀₀, m=12; 5B; PEI₆₀₀, m=3; 5C: PEI₆₀₀, m=8. This is ageneral procedure that serves as a model for other synthetic proceduresinvolving similar compounds, and may be used to synthesize a series ofdegradable cationic lipopolymers.

EXAMPLE 5

Cell culture: HEK 293T, 208F, HT 1080-EGFP and HeLa-EGFP cells aremaintained in DMEM containing 10% FBS, 100 units/ml penicillin and 100μg/ml streptomycin at 37° C., 5% CO₂ and 100% humidity condition. Inthis media the cells have a doubling time of about 20 hours and weresplit every 3-4 days to avoid confluency.

EXAMPLE 6

Plasmid DNA preparation: pEGFP-N1 plasmid was purchased from BD SciencesClontech company, encodes a red-shifted variant of wild-type GFP whichhas been optimized for brighter fluorescence and higher expression inmammalian cells. The GFP protein was controlled by immediate earlypromoter of CMV (P_(CMV IE)). The plasmids were amplified in DH5α E.coli and purified with Qiagen Plasmid Max Preparation Kit, and alwayshad an A260/A280 greater than 1.7.

EXAMPLE 7

In vitro transfection: 293 and 208F cells were plated in 96-well tissueculture plates (5×10⁴ cells/well for 293 cells and 1×10⁴ cells/well for208F cells) and incubated overnight in DMEM with 10% FBS. The precisemixing order of the plasmid-polymer complex is a critical parameter inthe outcome of the transfection. For each well, an aliquot of 7.5 μllipopolymer solution at different concentration was added into 7.5 μlDNA solution containing 0.6 μg of pEGFP-N1 plasmid and mixed completely.The DNA and lipopolymer mixture were incubated for 15 minutes at roomtemperature to allow for the formation of DNA-lipopolymer complexes. Thecomplexes were added each well and the cells were incubated at 37° C.,5% CO₂ for 24 hours. The EGFP gene transfection efficiency wasdetermined by GFP signal analysis. Lipofectamine were used as positivecontrols according to the protocol provided by manufacturer.

EXAMPLE 8

Observation of GFP signal: Green fluorescent signal in transfected cellsfrom Example 3 were observed under fluorescent microscope (Olympus,filter 520 nm). Cells were photographed using a 10× objective. Thepercent of cells with GFP signal in transfected cultures was determinedfrom counts of three fields for optimal cationic polymer amounts. Thetransfection efficiency of lipofectamine 2000 was about 60% in 293cells, efficiency of 5A, 5B and 5C was about 25%, 55% and 40%respectively, but the fluorescent density of 5A and 5C was slightlylower (see FIG. 1). The gene transfection efficiency of abovelipopolymers was also detected in 208F, in which the GFP positive cellswas about 40%, 45%, 25% and 50% respectively, after transfectionmediated by 5A, 5B, 5C and lipofectamine 2000 (see FIG. 2). Although thelipopolymers were not as good as lipofectamine 2000, the transfectionefficiency of 5B was very close to lipofectamine 2000, a leadingtransfection reagent in the market. The results indicated that thelipopolymers have a potential to be transfection reagents for variouscells lines.

In order to quantify the transfection efficiency, the relativefluorescent unit of transfected cells was determined by fluorescentmicroplate reader (FLX 800, Bio-TEK Instruments Co Ltd). The relativefluorescent unit (RFU) of lipofectamine 2000 transfected 293 cells was14596, about 2.3 times of 5B transfected cells (6318), while the RFU oflipofectamine 2000 and 5C transfected 208F cell was 2544 and 1954respectively, indicated that in different cells the transfection reagentshowed different performance and the new lipopolymers could have betterperformance in some cell lines (see FIG. 3).

EXAMPLE 9

SiRNA delivery study: SiRNA delivery efficiency was determined inHT1080-EGFP and HeLa EGFP cells, which originated from HT 1080 and HeLacells respectively with stable EGFP gene expression. The SiRNA targetingEGFP gene and luciferase gene was synthesized by Dharmacon Research Inc.siRNA targeting EGFP and luciferase gene were 21 bp double strand RNA,the sequence of sense strand of them were AAC GAG AAG CGC GAU CAC AUG(SEQ ID NO.: 2) and AAG UGC GCU GCU GGU GCC AAC (SEQ ID NO.: 3)respectively.

1.5×10⁴ HT1080-EGFP and HeLa-EGFP cells were planted in 96-well platefor each well at 24 h before transfection. For each well, an aliquot of7.5 μl lipopolymer solution at different concentration was added into7.5 μl DNA solution containing 2.0 pmol siRNA and mixed completely. TheDNA and lipopolymer mixture were incubated for 15 min at roomtemperature to allow for the formation of SiRNA-lipopolymer complexes.The complexes were added to each well and the cells were incubated at37° C., 5% CO₂ for 48 hrs. Lipofectamine were used as positive controlsaccording to the protocol provided by manufacturer. The siRNA deliveryefficiency was determined by GFP signal analysis.

In both HT-GFP and HeLa GFP cells the 5C showed very high siRNAdelivery, because the GFP signal was greatly inhibited after 5C mediatedsiRNA delivery. The GFP signal in 5C mediated siRNA delivered HT-GFP andHeLa-GFP cells was lower than lipofectamine 2000 mediated siRNA deliverycells. The results indicated that 5C had higher SIRNA deliveryefficiency than lipofectamine 2000 in both cell lines (FIG. 4 and FIG.5). On the other hand samples 5A and 5B also showed siRNA deliveryefficiency, even though the efficiency was lower than lipofectamine2000. The relative fluorescent unit was determined by fluorescentmicroplate reader, and the results showed relative fluorescent level ofthe 5C mediated siRNA delivery HT1080-EGFP and HeLa EGFP cells only27-28% of no delivery reagent group, lower than lipofectamine 2000medicated siRNA delivery cells, which showed about 33 to 46% relativefluorescent level as compare to no delivery reagent group. In otherwords, the inhibition efficiency of EGFP gene expression in 5C mediateddelivered cells was about 72-73%, much higher than lipofectamine 2000,which showed 54-67% of inhibition efficiency. The results indicated that5C had higher siRNA delivery efficiency than lipofectamine 2000.

EXAMPLE 10

Cytotoxicity Assays: Cytotoxicity of cationic gene carriers wasevaluated in mammalian cells using 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). 48 hoursafter siRNA delivery by the methods described in Example 9, 10 μl of MTTsolution (5.0 mg/ml in PBS, Sigma) was added to each well, and incubatedat 37° C. for 3 hrs. The medium was then removed and 200 μl DMSO wasadded into each well to dissolve the formazan crystals produced byliving cells. The absorbance of the solution was measured at 570 nm.Cell viabilities was calculated using the equation: Viability(%)={Abs_(570(sample))/AbS_(570 control)}. The control indicated no anyreagent or siRNA was added into cells. The results showed that thecytotoxicity of all 3 samples, 5A, 5B, and 5C, were very low, higherthan 95% cell survival after siRNA delivery at optimal condition, whilethe cell viability was about 65% after siRNA delivery by lipofectamine2000. Because HeLa cells were sensitive to the cytotoxicity oftransfection reagent, the results suggested that the new lipopolymershad very low cytotoxicity.

EXAMPLE 11

Specificity of lipopolymers in siRNA delivery: To evaluate whetherinhibition of GFP signal was caused by cytotoxicity or othernon-specific factors, the specificity of siRNA delivery was studied. TheSiRNA targeting GFP and Luc gene were delivered into HT-GFP cells by 5A,5B and 5C. The GFP signal was observed at 48 hrs after delivery. It wasfound that the 5B and 5A had low delivery efficiency, because there wasnot any change in GFP signal. 5C showed very high delivery efficiency,because 72% of GFP was inhibited at 48 hrs after SiRNA delivery(targeting GFP gene) when at optimal condition. On the other hand, whenSiRNA targeting luc gene was used, not any inhibition was found. Theresults indicated the GFP signal was specifically inhibited by SiRNAdelivery, instead of cytotoxicity caused inhibition.

EXAMPLE 12

Degradation study: In order to detect biodegradation, the lipopolymerswere diluted at opti MEM to final concentration of 320 μg/ml andincubated at 37° C. for 2, 4, 8, 24 hours respectively. The DNA bindingaffinities of samples were determined by FITC-labeled antisense oligo.100 μl polymers were added into 100 μl oligo solution (2 μmol/L) in 96well plate while vortexing and the mixture was incubated for 15 min.After that, the fluorescent was determined by fluorescent microplatereader (sensitivity=45). The relative fluorescent unit of free oligosolution (the oligo solution only mixed with 100 μl opti MEM solution)was about 5200 RFU, and PEI25K or PEI 600 was 1823 and 4350respectively. The results indicated that PEI 25K with high DNA bindingaffinity showed high inhibition on fluorescent (about 64% inhibition),while PEI 600 showed very inhibition on fluorescent unit (18%), becausePEI 600 usually showed very low DNA binding affinity. As for thecationic lipid based polymers, the inhibition efficiency on fluorescentwas about 65% to 70%, indicating the polymers had high DNA bindingaffinity.

After incubation at 37° C. in opti MEM for 24 h, both PEI 25K and PEI600showed no significant change in DNA binding affinity, because almost nochange was found in fluorescent signal as compare to original samples,indicated that PEI 25K was not degradable. However, after beingincubated at 37° C. for 8 h, the inhibition efficiency of 5A, 5B and 5Con fluorescent was gradually reduced to about 10%, lower then PEI 600.The results indicated that the lipopolymers were degradable in opti MEM.

EXAMPLE 13

Pentaethylenehexamine (PEHA) (43 mg, 0.19 mmol) (Aldrich) was weighedand placed in a small vial, and 2 ml of ethanol was added. After thePEHA completely dissolved, compound 6 (n×100 mg, n×0.19 mmol) wasquickly added into the PEHA solution while stirring. The reactionmixture was stirred for 2 hours at room temperature (25° C.). Then, thereaction mixture was neutralized by adding 1 ml of 4M HCl in ether. Thewhite precipitate was filtered, washed with ethanol, and dried at roomtemperature under reduced pressure. The obtained polymer 7 wascharacterized by NMR and agarose gel electrophoresis.

Other crosslinked, degradable cationic lipopolymers were prepared in asimilar manner by varying the ratio of PEHA to 6 as follows: 7A:PEHA/6=1/1; 7B: PEHA/6=2/1; 7C: PEHA/6=3/1; 7D: PEHA/6=4/1; 7E:PEHA/6=5/1; and 7F: PEHA/6=6/1.

EXAMPLE 14

Cell culture: HEK 293 cells and HeLa 705 cells were maintained in DMEMmedium containing 10% FBS, 100 units/ml penicillin and 100 μg/mlstreptomycin at 37° C., 5% CO₂ and 100% humidity conditions. CHO-AA8 lucwas cultured in MEM medium containing 10% FBS and antibiotics, othercondition are the same as 293 and HeLa 705 cells. The cells have adoubling time of about 20 hours and were split every 3-4 days to avoidconfluency.

Plasmid DNA preparation: pEGFP-N1 plasmid, purchased from BD SciencesClontech company, encodes a red-shifted variant of wild-type GFP whichhas been optimized for brighter fluorescence and higher expression inmammalian cells. The GFP protein was controlled by immediate earlypromoter of CMV (P_(CMV IE)). pCMV-luc plasmid was constructed in sameway. The plasmids were amplified in DH5α E. coli and purified withQiagen Plasmid Max Preparation Kit, and had an A260/A280 greater than1.7.

DNA binding of polymer 7: A series of samples of polymer 7 and DNA werediluted separately in opti MEM, then 10 μl polymer 7 solution was addedinto the DNA solution (20 μg/ml) while vortexing. The polymer/DNA ratioswere 32:1, 16:1, 8:1 and 10:1. The mixtures were incubated at roomtemperature for 15 min to form polymer/DNA complexes. After that, 5 μlDNA loading buffer was added into the complexes and 15 μl of mixture wasadded into 0.3% agarose gel for each well. The sample was subjected toelectrophoresis at 100V for about 30 min and the gel was visualized withUV light. The results of DNA binding affinity are shown in FIG. 10. Mostsamples showed lower DNA binding affinity. For examples, even whenpolymer/DNA ratio was 32:1, the DNA electrophoresis pattern of 7B, 7C,7D, 7E and 7F/DNA mixture was same as free DNA (polymer amount was 0).However, when polymer/DNA ratio was 32:1 and 16:1, the plasmid wasretarded by 7A, indicating sample 7A had DNA binding affinity.

EXAMPLE 15

In vitro gene transfection: 293 cells were plated in 96-well tissueculture plates (5×10⁴ cells/well) and incubated overnight in DMEM with10% FBS. For each well, an aliquot of 7.5 μl lipopolymer 7 solution atdifferent concentration was added into 7.5 μl DNA solution containing0.6 μg of pEGFP-N1 plasmid and mixed completely. The DNA and lipopolymer7 mixtures were incubated for 15 min at room temperature to allow forthe formation of DNA-lipopolymer complexes. The complexes were added toeach well and the cells were incubated at 37° C., 5% CO₂ for 24 hrs. TheEGFP gene transfection efficiency was determined by GFP signal analysis.Lipofectamine were used as positive controls according to the protocolprovided by manufacturer. Green fluorescent signal in transfected cellsfrom samples were observed under fluorescent microscope (Olympus, filter520 nm). Cells were photographed using a 10× objective. The percent ofcells with GFP signal in transfected cultures was determined from countsof three fields for optimal cationic polymer amounts.

About 24 h after transfection, about 28% of 7A transfected 293 cellsshowed transfection efficiency. The result indicated that lipoploymer 7Ais a transfection reagent (FIG. 11).

EXAMPLE 16

Antisense oligo delivery: Dr. Kole in University of Northern Carolinadeveloped luciferase 705 gene system for functional assay of antisensedelivery. In this system, human β-globin with mutation at 705 wasinserted into the sequence between luciferase cDNA. This plasmid wasintroduced into HeLa cell for stable gene expression, the cell line wastermed as HeLa luc705. Usually the cells exhibit low luciferaseactivity, because it expresses the wrong luciferase protein. However,antisense oligo binding to 705 sequences will block the wrong splicingsite and produce luciferase protein with biological activity. Luciferase705 is now used as functional model in antisense oligo delivery: higherluciferase activity indicates higher efficiency of antisense delivery.

Antisense oligo delivery efficiency of polymer 7 samples was evaluatedin Luc 705 cell line. The final concentration of oligo targeting luc 705was 1.0 μmol/L, the concentration of polymers was 320 and 160 μg/ml(same as the amount of polymers when polymer/DNA ratio was 16: and 8:1during transfection. The luciferase activity was determined) about 24 hafter transfection.

The background luciferase activity in 705 cell was about 1.6×10⁵ RLU/mgprotein. The luciferase activity of antisense oligo delivered to thecell by 7A was greatly increased, near 50 times that in thenon-delivered cells. The delivery efficiency of lipofectamine 2000 wasless, about 15 times that in the non-delivered cells. The resultsindicated that lipid-polymer 7A has higher antisense oligo deliveryefficiency than lipofectamine 2000 (FIG. 12).

EXAMPLE 17

The SiRNA delivery efficiency of lipo-polymers was determined inCHO-AA8-luc cell line. CHO-AA8 Luc is Tet-off cell lines, in whichluciferase gene expression was controlled by Dox. The luciferase geneexpression will be shut down, when Dox is added into cell, and it willexpress after Dox is removed.

In SiRNA delivery study, the existing protein of a target gene mayaffect the evaluation, because if the half-life time is long, theprotein may be detected, even though the target gene was already shutdown. In this case, CHO-AA8 Luc cell showed advantages. The luciferasegene expression could be shut down by adding Dox to reduce backgroundprotein. During SiRNA delivery, the Dox could be removed by changing themedium, and the luciferase gene began to express, at same time, if theSiRNA was successfully delivered into the cell, the luciferaseexpression level could be inhibited by SiRNA.

The CHO AA8 luc was seeded in 96 well plate with Dox. 18-24 h later, theSiRNA cassette/polymer 7 complexes were made. The final concentration ofpolymers was 320 and 160 μg/ml (same concentration as that 16:1 and 8:1in gene transfection). The amount of SiRNA cassette was 150 ng/ml. Aftermedium change and the cell washed with PBS, the SiRNA/polymer 7complexes were added into cell. After cell was incubated at 37° C. for48 h, luciferase activity was detected.

The luciferase activity of control (blank) was about 10⁷ RLU/mg protein,after SiRNA cassette delivery by 7A and lipofectamine 2000, theluciferase activity was greatly inhibited to 2.47 and 3.49×10⁶ RLU/mgprotein respectively. The results indicated 7A could efficientlydelivery siRNA cassette into cells and the delivery efficiency washigher than lipofectamine (FIG. 13).

EXAMPLE 18

Cytotoxicity of cationic lipopolymer 7 gene carriers were evaluated inmammalian cells using 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). 48 h aftergene transfection by the methods described above, 10 μl of MTT solution(5.0 mg/ml in PBS, Sigma) was added to each well, and incubated at 37°C. for 3 hours. The medium was then removed and 200 μl DMSO was addedinto each well to dissolve the formazan crystals produced by livingcells. The absorbance of the solution was measured at 570 nm. Cellviabilities was calculated using the equation: Viability(%)={Abs_(570 (sample))/Abs_(570 control)}. The results showed that thecytotoxicity of 7A was very low, more than 95% cell survival after siRNAdelivery at optimal condition, while the cell viability was about 75%after siRNA delivery by lipofectamine 2000. The results indicated 7A hasvery lower cytotoxicity (FIG. 14).

EXAMPLE 19

Biodegradation of polymer 7: Polymer 7A was diluted with opti MEM to afinal concentration 320 μl/ml. A series of samples were incubated at 37°C. for 4 hours, 8 hours and 24 hours, then samples were taken for genetransfection study in 293 cell seed in 96 well plate. The transfectionwas performed by the protocol described above. The GFP signal wasobserved at 24 hours after transfection.

The GFP gene transfection efficiency of 7A was about 25%, and there wasno significant change in transfection efficiency after the samples wasincubated for 4 hours. The transfection efficiency was greatly reducedafter the sample was incubated for 8 h, and almost no transfectionefficiency was found after the sample was incubated for 24 hours. Theresults indicated that the 7A was biodegradable under neutral conditions(FIG. 15).

It will be appreciated by those skilled in the art that variousomissions, additions and modifications may be made to the compositionsand methods described above without departing from the scope of theinvention, and all such modifications and changes are intended to fallwithin the scope of the invention, as defined by the appended claims.

1. A polymer comprising a recurring unit of formula (Ia):

wherein: PEI is a polyethyleneimine recurring unit; R is an electronpair or hydrogen; L is selected from the group consisting of a C₁₂ toC₁₈ fatty acid, cholesterol, a C₁₂ to C₁₈ fatty acid derivative and acholesterol derivative; and m is an integer in the range of about 1 toabout
 30. 2. The polymer of claim 1, wherein the PEI is represented by arecurring unit of formula (II)

wherein x is an integer in the range of about 1 to about 100 and y is aninteger in the range of about 1 to about
 100. 3. The polymer of claim 1,wherein the polyethyleneimine recurring unit has a molecular weight inthe range of about 600 Daltons to about 25,000 Daltons.
 4. The polymerof claim 3, wherein the polyethyleneimine recurring unit has a molecularweight of about 600 Daltons.
 5. The polymer of claim 4, wherein L is aC₁₂ to C₁₈ fatty acid or a C₁₂ to C₁₈ fatty acid derivative.
 6. Thepolymer of claim 1, wherein L is a C₁₂ to C₁₈ fatty acid or a C₁₂ to C₁₈fatty acid derivative.
 7. The polymer of claim 6, wherein L has thestructure:


8. The polymer of claim 1, wherein the polymer is biodegradable.
 9. Thepolymer of claim 1, wherein the polymer degrades by a mechanism selectedfrom the group consisting of hydrolysis, enzyme cleavage, reduction,photo-cleavage, and sonication.
 10. The polymer of claim 1, having aweight average molecular weight in the range of about 500 Daltons toabout 1,000,000 Daltons.
 11. The polymer of claim 1 having a weightaverage molecular weight in the range of about 2,000 Daltons to about200,000 Daltons.
 12. The polymer of claim 1, wherein the polymer iscrosslinked.
 13. A composition comprising a biomolecule that iscomplexed to the polymer of claim
 1. 14. The composition of claim 13,wherein the biomolecule is a nucleic acid.
 15. The composition of claim14, wherein the nucleic acid is siRNA.
 16. The composition of claim 13,further comprising a delivery enhancing agent capable of entering aeukaryotic cell.
 17. The composition of claim 16, wherein the deliveryenhancing agent facilitates one or more functions in the eukaryotic cellselected from the group consisting of receptor recognition,internalization, escape of the biomolecule from cell endosome, nucleuslocalization, and biomolecule release.
 18. A pharmaceutical compositioncomprising the polymer of claim
 1. 19. The pharmaceutical composition ofclaim 18, further comprising a sensitizer agent.
 20. The pharmaceuticalcomposition of claim 19, wherein the sensitizer agent is sensitive tovisible radiation, ultraviolet radiation, or both.
 21. A method ofdelivering a biomolecule to a eukaryotic cell, comprising contacting thecell with the composition of claim 13 to thereby deliver the biomoleculeto the cell.
 22. A method of administering a nucleic acid to a mammalcomprising locally administering the composition of claim 14 to a cellof said mammal.